Radiolabeled vasoactive intestinal peptide analogs for imaging and therapy

ABSTRACT

This invention relates to a radiodiagnostic agent to image tumors. A composition for a tumor imaging agent, a method and kit for preparing a tumor imaging agent, and a radiolabeling reagent for preparing the tumor imaging agent are provided. Methods of using the tumor imaging agent to detect tumors are also provided

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. Non-provisionalpatent application Ser. No. 09/333,842, filed Jun. 15, 1999, now U.S.Pat. No. 6,395,255, which claims priority based under 35 U.S.C. §119based upon U.S. Provisional Patent Application No. 60/089,364, filedJun. 15, 1998.

FIELD OF THE INVENTION

This invention relates generally to the field of nuclear medicine and,more particularly to, radiolabeled agents for imaging tumors, methods ofuse of said agents, and kits for preparing imaging agents.

BACKGROUND OF THE INVENTION

Cancer remains a major health problem among humans. In the United Statescolorectal cancer is the second most common cancer in women after breastcancer, and the third most common cancer in men after prostate and lungcancer. While the colorectal cancer incidence rates for white and blackmales are similar (roughly 61 per 100,000 population), the rate forwhite females (88 per 100,000 population) is higher: than that for blackfemales (79 per 100,000 population). However, the mortality rate forboth black males and black females is higher than their whitecounterparts, suggesting that late diagnosis may contribute to themortality rate. Moreover, in that the incidence and mortality ratesrelated to this disease continue to rise, the need for early diagnosisis of undisputed importance for management of the cancer. (Scottenfeld,D., Cancer of the Colon, Rectum and Anus, Chapter 3, Epidemiology,Cohen, A. (Ed.), McGraw Hill, Inc., New York, 1993, pp. 11-24). If thediagnosis is made while the cancer is still silent or occult, the cancermay be treated with conservative surgery, with or without additive,radio- or chemotherapy.

During the past 15 years, advances have been made in diagnostic imagingof tumors. The introduction of computerized tomography (CT) byHounsfield in the early 1970's, enabled more accurate detection withhigher spatial resolution for small foci of cancer than previous x-raytechnology. Similarly, magnetic resonance imaging (MRI) improved thediagnostic capabilities for detecting bone malignancies.

Gamma cameras have generally been used for diagnostic purposes becausethey are considerably less expensive than either CT or MRI scanners, andthe scintigraphic studies are more cost effective than the CT or MRIimaging. Gamma cameras enable a patient's entire body to be scanned atone time and in a relatively short period, allowing a single diagnostictest to be utilized for all imaging needs. However, while nuclearmedicine can facilitate the diagnosis of cancer, there remains a needfor better imaging techniques and more specific imaging agents fordetecting tumors, particularly those that have metastasized.

Recently, CT, MRI and ultrasound imaging have been criticized for thedetection of colorectal adenocarcinoma. (Beutow, P. C. et al.,Radiographics 15: 127-146, 1995). The manner of routine screening forthis disease remains controversial, and it is generally agreed that CTshould not be used routinely to stage colorectal carcinoma because ofits low accuracy. In that the five year survival rate patients withcolorectal cancer is only 5%, and that this rate has not changed for thepast 40 years, it is evident that the early detection of small cancersand pre-malignant adenomas is necessary to improve the survival rate.For early detection to be practical, new non-invasive methods ofdetection of this disease are necessary.

Biomolecules specific for tumor antigens, when labeled with gammaemitting radionuclides, can provide an efficient means of detectinglesions. Oncoscint, an In-111 labeled anti-CEA and anti-TAG-72 antibody,has been utilized as a tumor imaging agent. However, this agent has hadonly limited success because of relatively low sensitivity (40%) andspecificity (50%) (Beatty, J. D. et al., Cancer of the Colon, Rectum andAnus, Radioimmunoscintigraphy, Chapter 77, Cohen, A. (Ed.), McGraw Hill,Inc., New York, 1993, pp. 753-767; John, T. M. et al., Proc. Annu. Mtg.Am. Soc. Clin. Oncol. 13: A275, 1994), and because it induces animmunologic reaction in 30% of the patients. (Corman, M. L. et al.,Diseases of the Colon and Rectum 37: 129-137, 1994). The imaging agentis also of limited because of its high liver uptake which precludes itsuse for detecting liver metastases. (Abdel-Nabvi, H. H. et al., TargetedDiag. and Therapy 6: 78-88, 1992). Agents with improved specificity andsensitivity, and ones that can be used reliably to detect livermetastases would be widely accepted by clinicians.

Radiolabeled receptor peptides specific for imaging tumors, abscessesand vascular thrombi are particularly attractive for use as radioimagingagents because they are smaller in size, easier to produce, and clearedmore rapidly from the blood than other receptor specific largermolecules, such as radiolabeled monoclonal antibodies.In-111-Octreotide, a radiolabeled somatostatin analog, has been used toimage endocrine tumors. However, it does not appear that this imagingagent is as sensitive as an I-123 labeled vasoactive intestinal peptide(VIP). Only four positive scans were obtained in 17 patients withcolonic adenocarcinoma using In-111-Octreotide, as compared to 17 out of17 positive scans with I-123 labeled VIP. (Virgolini, I. et al., NewEng. J. of Med. 33: 1116-21, 1994).

VIP is a 28 amino acid neuroendocrine mediator that has been detected onthe cell surface membrane of intestinal epithelial cells (Virgolini, I.et al., Cancer Res. 54: 690-700, 1994; Blum, A. M. et al., J.Neuroimmunol. 39:101-8, 1992), lungs (Couvineau, A. et al., J. Clin.Endocrinol., Metab. 61: 50-55, 1985), and various tumor cells, includingcolonic adenocarcinomas (el Battari, A. et al., J. Biol. Chem. 263:17685-9, 1988), pancreatic carcinomas (Svoboda, M. et al., Eur. J.Biochem 176: 707-13, 1988) and carcinoids (Virgolini, I. et al., CancerRes. 54: 690-700, 1994). The amino acid sequence for VIP, which isidentical for porcine, bovine, dog and human, is His Ser Asp Ala Val PheThr Asp Asn Tyr Thr Arg Leu Arg Lys Gln Met Ala Val Lys Lys Tyr Leu AsnSer Ile Leu Asn (SEQ ID NO: 1). It was initially isolated from porcineintestine more than 25 years ago. (Said, et al., Science 69: 1217-1218,1970).

When labeled with I-123, VIP has been shown to be effective in imagingcolorectal adenocarcinoma (100%), including in patients with livermetastases (83%). However, the lung uptake of this agent is very high:approximately 25% at four hours post-injection and 10% at 24 hourspost-injection. It is unclear at present whether or not the high lunguptake is related to lung receptor density or to iodination of the twotyrosine residues (residues 10 and 22 of VIP). Accordingly, iodinatedVIP adversely affects imaging of lung metastases (66%).

Further, although I-123 is widely used for scintigraphic imaging, andmay be a logical choice to label a tyrosine containing peptide, it is acyclotron produced radionuclide with a relatively short half-life (13.3hours). It is therefore expensive to produce, and must be ordered within24 hours of its intended use to prevent excessive radioactivity decay.For these reasons I-123 has not been widely utilized for clinicalapplications.

Conversely, Technetium-99m (“Tc-99m”), is produced on a generator fromits parent radionuclide Molydenum-99 (“Mo-99”), which has asubstantially longer half-life (2.8 days). Mo99, bound on solid matrixin several mCi quantities, can be purchased once a week. The Tc-99m iswashed off with sterile isotonic saline once or twice a day as needed.Tc-99m decays within a six hour half-life with the emission of 140 keVgamma rays. The shorter half-life permits rapid decay of theradioactivity, minimizes the radiation dose to normal organs, andeliminates the need for patient hospitalization. Its gamma ray energyalso allows efficient detection by gamma cameras. Further, because ofthe generator system, the radionuclide is relatively inexpensive. Forthese reasons Tc-99m has become the radionuclide of choice in nearly 90%of the clinical nuclear medicine applications.

It has now been found that a modification of the peptide sequence HisSer Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln Met Ala ValLys Lys Tyr Leu Asn Ser Ile Leu Asn (SEQ ID NO: 1) can be used to imagetumors, particularly colonic adenocarcinomas, liver metastases,pancreatic carcinomas, and carcinoids.

SUMMARY OF THE INVENTION

The present invention provides a composition useful as a radiodiagnosticagent for imaging tumors, particularly endocrine tumors, livermetastases, and carcinoids in mammals, a method and a kit for thepreparation of the tumor imaging agent, a reagent for radiolabeling theimaging agent, and a method of use for the tumor imaging agent.Specifically, the tumor imaging agent is comprised of a compositioncontaining a tumor specific sequence (TSS) and a radionuclide moietylinked to the TSS via a linker, wherein a radionuclide is complexed tothe radiolabeling moiety. In a preferred embodiment, the compositioncomprises the sequence His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr ArgLeu Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn Xaa GlyGly (D)Ala Gly (SEQ ID NO: 2).

A second embodiment of the invention provides a reagent forradiolabeling a TSS, comprising four amino acids, selected from thegroup consisting of glycine and alanine, which can covalently link aselected radionuclide to the amino groups of each amino acid to form anN₄ configuration.

Still another embodiment of the invention provides a kit for preparing athrombus imaging agent, which kit comprises a container capable ofholding a multiple of vials and reagents. A first vial contains anappropriate quantity of prepared TSS and radionuclide moiety forreacting with a radionuclide. A second vial contains an appropriatebuffer,

The invention also provides a method of imaging endocrine tumors in amammal by obtaining in vivo gamma scintigraphic images. The methodcomprises administering an effective diagnostic amount of the thrombusimaging agent to a mammal in need of such imaging and detecting thegamma radiation emitted by the imaging agent localized at the thrombussite within the mammal.

DESCRIPTION OF THE FIGURES

FIG. 1 is a structural representation of a tumor imaging agent.

FIG. 2 is the HPLC elution profile of ^(99m)Tc-TP3654. One hundredpercent radioactivity is eluted in single peak at Rt 11.8 min. This isconsiderable improvement over multiple peaks with CPTA-VIP and MAG₃-VIPanalogs. A diagonal line represents gradient composition.

FIG. 3 is a graph of the effect of increasing concentration of VIP₂₈ ,unlabeled TP3654 and CPTA-VIP on resting IAS tension. At 10⁻⁶ mol/LTP3654, 95% muscle relativity was achieved, whereas relativity wasapproximately 75% with VIP₂₈ and 35% with CPTA-VIP at the sameconcentration.

FIG. 4 is a graph of ¹²⁵I-VIP displacement curves with unlabeled TP3654and VIP₂₈.

FIG. 5 consists of posterior gamma camera images of nude mice bearingLS174T human colorectal tumor in the right thigh. Both images wereobtained 24 h after injection of ^(99m)Tc-TP3654 (left) and^(99m)Tc-G(D)AGG-Aba (right). Tissue distribution is distinctlydifferent. Despite marginal uptake (0.2% ID/g), tumor in the right thighis delineated with ^(99m)Tc-TP3654 because of low body background. Thistumor is not delineated with ^(99m)Tc-G(D)AGG-Aba. Both tumors weighapproximately 1 g.

FIG. 6 is a blood clearance curve demonstrating a biphasic behavior of^(99m)Tc-TP3654. The α-t_(1/2) was approximately 5 min and the β-t_(1/2)was approximately 120 min.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions

1. Abbreviations

Aba 4-aminobutyric acid ADP Adenosine 5′-diphosphate CPTA (4-(1, 4, 8,11-tetraazacyclotetradec-1-yl)methyl) benzoic acid LS174T Humancolorectal tumor cell line MAG₃(N-(N(N-(benzylthio)acetyl)glycyl)glycyl)glycin TFA Trifluoroacetic acidTSS Tumor specific sequence VIP Vasoactive intestinal peptide Xaa4-aminobutyric acid

2. Definitions

“Natural amino acid” means any of the twenty primary, naturallyoccurring amino acids which typically form peptides and polypeptides. By“synthetic amino acid” is meant any other amino acid, regardless ofwhether it is prepared synthetically or derived from a natural source.

“Radiolabeling moiety” is a sequence comprising four amino acids capableof complexing with a selected radionuclide in an N₄ configuration.

“Radiolabeled complex” is the complex formed by the radiolabeling moietyand the selected radionuclide.

“Technetium” refers to the radioactive form of technetium, for example,technetium-99m (Tc-99m).

“Tumor imaging agent” refers to a radiolabeled TSS to be used as aradiodiagnostic agent.

“Tumor specific sequence (“TSS”)” refers to a peptide with the sequenceHis Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln Met AlaVal Lys Lys Tyr Leu Asn Ser Ile Leu Asn (SEQ ID NO: 1) or an analog orfragment thereof, to which one or more natural or synthetic amino acidsand a linker have been added to the NH₂ or COOH termini.

Tumor Imaging Agents

This invention is directed to a radiodiagnostic agent to image tumors ina mammal. The invention provides a composition useful as a tumor imagingagent, a method and kit for preparing the tumor imaging agent, and aradiolabeling reagent for preparing the tumor imaging agent. Tumors asdescribed refers to tumors that express the VIP receptor, including, butnot limited to, breast, ovarian, endometrial, prostate, bladder, lung,esophageal, colonic and pancreatic cancers, and neuroendocrine and braintumors.

The tumor imaging agent is a composition for imaging tumors in a mammalhaving the formula of I or II:

M-Z-X₁—P—X₂  (I)

X₁—P—X₂-Z-M  (II)

wherein:

M is a radiolabeling moiety comprised of four amino acids capable ofcomplexing with a selected radionuclide in an N₄ configuration;

Z is a linker comprised of one or more natural or synthetic amino acids;

X₁ and X₂ are from zero to twenty natural or synthetic amino acids; and

P is a peptide comprising the sequence His Ser Asp Ala Val Phe Thr AspAsm Tyr Thr Arg Leu Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser IleLeu Asn (SEQ ID NO: 1), or an analog or fragment thereof,

wherein a radionuclide is complexed to the radiolabeling moiety, M ofthe composition of formula (I) or (II). In a preferred embodiment thecomposition comprises His Ser Asp Ala Val Phe Thr Asp Asm Tyr Thr ArgLeu Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn Xaa Gly(D)Ala Gly Gly (SEQ ID NO: 2) or analog or fragment thereof.

The tumor imaging agent is comprised of a TSS and a radiolabeled complexconnected by the linker of the TSS. By “TSS” it is meant a peptide withthe sequence, His Ser Asp Ala Val Phe Thr Asp Asm Tyr Thr Arg Leu ArgLys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn (SEQ ID NO: 1)or an analog or fragment thereof, to which one or more natural orsynthetic amino acids and a linker have been added to the NH₂ and/orCOOH termini. The linker comprises one or more natural or syntheticamino acids that will preserve the biological activity of the peptidesequence from any steric hindrance imparted by the use of theradionuclide moiety. Preferably, the TSS is comprised of from about 28to about 50 amino acids, more preferably from about 28 to about 40 aminoacids, and most preferably, from about 28 to about 30 amino acids.

In a preferred embodiment the linker, 4-aminobutyric acid Xaa, has beenadded to the COOH terminus of a sequence, such as SEQ ID NO: 2.Alternatively, one or more amino acids and a linker could be added tothe NH₂ terminus.

Analogs of the peptide of the TSS can also be utilized in the invention.By “analog” is meant a derivative or modification of the nativesequence. One skilled in the art may prepare such analogs wherein thenative sequence is modified by resultant single or multiple amino acidsubstitutions, additions or deletions. All such modifications resultingin a derivative TSS are included within the scope of the invention,provided that the molecule binds to the tumor and thereby, acts as atumor imaging agent when radiolabeled.

Conservative amino acid changes may be made which do not alter thebiological function of the native sequence. For instance, one polaramino acid, such as threonine, may be substituted for another polaramino acid, such as serine; or one acidic amino acid, such as asparticacid, may be substituted for another acidic amino acid, such a glutamicacid; or a basic amino acid, such as lysine, arginine or histidine, maybe substituted for another basic amino acid; or a non-polar amino acid,such as alanine, leucine or isoleucine, may be substituted for anothernon-polar amino acid.

Accordingly, an analog as described herein corresponds not only to thenative peptide described herein, but also to any analog or fragmentthereof which retains the essential biological activity of the peptide.Analogs include any peptide having an amino acid sequence substantiallysimilar to that of SEQ ID NO: 2 in which one or more amino acids havebeen substituted or inserted in the native sequence. Fragments includepeptides of the length less than the full length of SEQ ID NO: 2. Thepractice of the present invention is, thus, not limited to sequences ofthe same length as SEQ ID NO: 2, but also includes such fragments ofthis TSS, provided they bind to the tumor when complexed with theradiolabel moiety and thus, act when radiolabeled as a tumor imagingagent. Whether an analog or fragment retains the biological activity ofthe native TSS may be determined by those skilled in the art byfollowing the experimental protocols set forth in herein.

Another embodiment of the invention is the preparation of aradiolabeling moiety for use as a reagent in the production of athrombus imaging agent. The radiolabeling moiety is capable ofcomplexing with a radionuclide in an N₄ configuration. An N₄configuration is one in which the radionuclide is linked to each aminoacid through its NH₂ group. While the N₄ configuration is a preferredembodiment for the present invention, other configurations are includedwithin the practice of the present invention. Thus, radiolabeledcomplexes in which a radionuclide such as technetium is joined to asingle thiol moiety or two amino and two thiol moieties (N₂S₂configuration) are equivalent to the N₄ configuration.

For the preparation of the radiolabeled complex as described herein,four amino acids (the radiolabeling moiety) are covalently linked (orcomplexed) to a selected radionuclide. Preferably, the amino acids oreindependently selected from the group consisting of glycine and alanine,provided that at least three of the amino acids are glycine.Alternatively, any combination of amino acids that form an N₄configuration can be utilized within the scope of this invention. Aminoacids of both the D and L enantiomeric configuration can be employedherein. Naturally occurring or synthetic amino acids can be employedherein. In a preferred embodiment, the sequence for the radiolabelingmoiety is Gly (D)Ala Gly Gly (SEQ ID NO: 3).

In a preferred embodiment technetium-99m is selected as theradionuclide. However, examples of other suitable radionuclides whichcan be complexed with this moiety include, but are not limited to,Re-186, Re-188, In-111, Ga-67, Ga-68, Tl-201, Fe-52, Pb-203, Co-58,Cu-64, I-123, I-124, I-125, I-131, At-210, Br-76, Br-77 and F-18.

Another embodiment of the invention is the use of the tumor imagingagent to image tumors in mammals, preferably humans. A protocol for suchuse is provided in Example. Tumor imaging agents of the invention areadministered to a mammal in need of such imaging, i.e., suspected ofhaving a tumor, by intravenous injection. The tumor imaging agent isadministered in a single unit injectable dose at a concentration whichis effective for diagnostic purposes. The tumor imaging agent isadministered intravenously in any conventional medium, such as isotonicsaline, blood plasma, or biologically compatible isotonic buffers, suchas phosphate, Hepes or Tyrode's buffer. Generally, the unit dose to beadministered has a radioactivity of about 0.01 to about 100 mCi,preferably about 1 to 40 mCi. The solution amount to be injected as aunit dose is from about 0.1 ml to about 50.0 ml. Preferably, the amountinjected is from about 0.5 to about 5 ml (same thing). Imaging of thetumor can take place within a few minutes of injection. However, imagingcan take place, if desired, several hours after injection. In mostinstances, a sufficient amount of the administered dose will accumulatein the desired area within a few minutes to a few hours after injectionto permit the taking of scintigraphy images. This is an “effectivediagnostic amount”. Any conventional method of scintigraphic imaging,planar, SPECT or PET, for diagnostic purposes, can be utilized inaccordance with this invention.

Still another embodiment of the invention is a kit for the preparationof the tumor imaging agent. An example of such a kit is provided inExample. The kit includes a carrier for holding the kit components andcontainers of the TSS, reducing agent and buffer.

The methods for making and using the tumor imaging agent of theinvention are more fully illustrated in the following examples. Theseexamples illustrate certain aspects of the above-described invention andare shown by way of illumination and not by way of limitation.

EXAMPLES

Preparation of Tumor Imaging Agent

The tumor imaging agent of the invention described herein was preparedaccording to the following protocols.

Preparation of Composition of Formula (I) or (II)

The composition corresponding to the peptide His Ser Asp Ala Val Phe ThrAsp Asm Tyr Thr Arg Leu Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn SerIle Leu Asn Xaa Gly (D)Ala Gly Gly (SEQ ID NO: 2) was prepared usingWang resin and an automated synthesizer (Applied Biosystems Inc., ModelNo. 430). The peptide was cleaved from the resin with 90% TFA andprecipitated by the addition of diethylether at −20° C. The peptide waspurified using preparative high-performance liquid chromatography (HPLC;Shimadzu LC-10 AD Columbia, Md.) and a 5-• C₁₈ HAlsil column. Fractionswere collected and lyophilized, and the resultant compounds werecharacterized using a Perkin Sciex APZ ion-spray mass spectrometer(Norwalk, Conn.). The peptide with the linker and the four amino acidscomprising the radiolabeling moiety had the observed molecular weight of3654.48, as compared to the theoretically expected weight of 3654.32.This analog is referred to as TP3654. The chemical aspects of thispreparation have been described elsewhere, the details of which areincorporated by reference herein. (Pallela et al., J Nucl Med. 39: 226P,1998).

Preparation of ¹²⁵I-VIP and ¹²⁵I-TP3654

Both VIP and TP3654 contain two tyrosine residues (positions 10 and 22)and facilitate radioiodination, as demonstrated previously. (Virgoliniet al., N Engl J Med. 331:1116-1121, 1994). In brief, 100 •g iodogen in10 •L CHCl₃ was placed in a clean, siliconized, conical glass vial, andchloroform was evaporated by a gentle stream of nitrogen. Then, 10 •gVIP or TP3654 in 10 •L 0.5 mol/L phosphate buffer, ph 7.5, was added,followed by approximately 1 mCi (¹²⁵I)NaI solution in approximately 100•L 0.5 mol/L phosphate buffer, pH 7.5. The vial was sealed, and thesolution was mixed in a vortex mixer and allowed to react for 30 min at22° C. The reaction was terminated by adding 500 •g sodiummetabisulfite. The product was purified using a Rainin 4.6 mm×25 cm,C₁₈(5-•) microbond reverse-phase column (Woburn, Mass.) connected to aRainin HPLC equipped with NaI (Tl) radioactivity detector and anultraviolet monitor. The eluting solvent consisted of 0.1% TFA in water(solvent A) and 0.1% TFA in acetonitrile (solvent B) at a flow rate of 1mL/min. The mono-iodinated VIP or TP3654 was collected and solventevaporated, and then the ¹²⁵I-VIP or ¹²⁵I-TP3654 was taken up in asuitable volume of 0.1 mol/L tris(hydroxymethyl)aminomethane (Tris)buffer, pH 10, containing 0.1% human serum albumin (HSA) and stored at−80° C. On one occasion, ¹²⁵I-VIP was purchased from Amersham LifeSciences (Buckinghamshire, UK).

Preparation of ^(99m)Tc-TP3654

To a clean, nitrogen-flushed, 10-mL glass vial was added 50 •g TP3654 inacetate buffer (pH 4.6), 50 •g SnCl₂×H₂O in 10 •L 0.005 mol/L HCl and300 •L 0.1 mol/L trisodium phosphate (pH 12.0). The content was frozenimmediately by placing the vial in an acetone dry-ice bath. The vial wasthen placed in a GeneVac lyophilizer (Sheffield, UK) and was lyophilizedfor 2 h. The vials were then filled with nitrogen, sealed and stored at−20° C.

To a vial at 22° C., 10-40 mCi ^(99m)Tc in 0.1-0.6 mL 0.9% NaCl wasadded and mixed using a vortex mixer. The mixture was incubated for 15min. Then, the pH of the reaction mixture was raised to 6-6.5 by theaddition of 1-1.5 mL 0.1 mol/L Na₂HPO₄ solution, pH 5.2. Ascorbic acid(500 •g) was then added as a stabilizing agent. HPLC analysis wasperformed using a Rainin HPLC with a reverse-phase C₁₈ microbond columnand with 0.1% TFA in H₂O (solvent A) and 0.1.% TFA in acetonitrile(solvent B). The gradient was such that solvent B was 10% at 0 min and90% at 28 min. In instant thin-layer chromatography (ITLC-SG; GelmanSciences, Ann Arbor, Mich.) using pyridine:acetic acid:water (3:5:1.5)as a mobile phase, colloid remains at Rf 0.0 and ^(99m)Tc-TP3654migrates at Rf 1.0.

Stability of ^(99m)Tc-TP3654

To measure the in vitro stability of ^(99m)Tc-TP3654, a known quantityof the preparation was incubated at 37° C. for 24 h, with 100 molarexcess of DTPA, HSA or cysteine. At 0, 5 and 24 h, samples werewithdrawn for HPLC analysis and the radioactivity that was associatedwith the challenging agent and that remained as ^(99m)Tc-TP3654 (Rt=11.8min) was measured.

Functional Assay Using Opossum Anal Sphincter Smooth Muscle Tissues

This assay was performed by using the method of Rattan and colleagues(Chakder, S. et al., J Pharm Exp Therapeut. 266:392-399, 1993; Moummi,C. et al., Am J Physiol. 1988;255:G571-G578) to examine the biologicalactivity of TP3654 and CPTA-VIP. Native VIP₂₈ was used as a control.Multiple lines of evidence suggest that VIP is on eof the inhibitoryneurotransmitters in the gut. The assay is based on the binding of VIPto specific receptors that cause a decrease in the resting tension ofthe internal anal sphincter (IAS) smooth muscle. This was determined inthe presence of increasing concentrations of VIP until maximum fall wasreached.

Preparation of Smooth Muscle Strips

Adult opossums (Didelphis virginiana) of either sex were anesthetizedwith pentobarbital (40 mg/kg intraperitoneally) and then were killed.The anal canal was removed and was transferred quickly to oxygenatedKrebs' physiological solution of the following composition (in mmol/L):118.07 NaCl, 4.69 KCl, 2.52 CaCl₂, 1.16 MgSO₄, 1.01 NaH₂PO₄, 25 NaHCO₃and 11.10 glucose. The large blood vessels and extraneous tissues thatcontained the external anal sphincter were removed by sharp dissection,and the anal canal was opened and pinned flat with the mucosal side upon a dissecting tray containing oxygenated Krebs' physiologicalsolution. The mucosa was remove by using forceps and fine scissors, andthe IAS circular smooth muscle strips (approximately 2 mm wide and 1 cmlong) were cut from the lowermost part of the anal canal. Silk suturewas tied to both ends of these muscle strips for isometric tensionmeasurements.

Measurement of Isometric Tension

The IAS smooth muscle strips were transferred to temperature-controlled2-mL muscle baths containing Krebs' solution bubbled continuously with amixture of 95% O₂ and 5% CO₂.

The lower end of the muscle strip was tied to the bottom of the musclebath with the tissue holder, and the other end was attached to anisometric force transducer (model FTO3; Grass Instruments Co., Quincy,Mass.). Isometric tensions of the smooth muscle strips were recorded ona Beckman Dynograph recorder (Beckman Instruments, Schiller Park, Ill.).Initially, 1 g of tension was applied to the muscle strips, which werethen allowed to equilibrate for about 1 h with occasional washings.During the equilibration period, strips developed steady tension. Onlystrips that developed steady tension and relaxed in response toelectrical field stimulation were used. Both optimal length and baseline of the muscle strips were determined, as described previously.(Chakder, S. et al., J Pharm Exp Therapeut. 266:392-399, 1993; Moummi,C. et al., Am J Physiol. 1988;255:G571-578).

Drug Responses.

TP3654 and CPTA-VIP were chosen as test agents and VIP₂₈ as a control.The effect of different concentrations of these agents on resting IAStension was examined using cumulative concentration responses. After agiven concentration-response curve was derived, the muscle strips werewashed continuously for 45-60 min before testing for theconcentration-response curve of another agent. Maximal relaxation (100%)of the smooth muscle strips was determined after completely relaxing themuscle strips with 5 mmol/L ethylenediaminetetraacetic acid (EDTA).

¹²⁵I-VIP Receptor Displacement Assay with TP3654 and VIP

Colon adenocarcinoma cell line HT-29 was purchased from American TypeCulture Collection (ATCC (Manassas, Va.)) and maintained in McCoy's 5Amedium containing 10% heat-inactivated fetal bovine serum (FBS), 1%penicillin-streptomycin and 1% minimum essential medium (MEM) vitamins(Mediatech Inc., Herndon, Va.) at 5% CO₂ and 37° C.

Confluent cells were collected, washed with 50 mmol/L Tris×HCl buffer(pH 7.5) and then resuspended in 50 mmol/L Tris×HCl buffer (pH 7.5) thatcontained 5 mmol/L MgCl₂, 1 mmol/L CaCl₂ and 0.15 mol/L NaCl at 4° C.Approximately 2.5×10⁶ viable cells were dispensed, in triplicate, inseveral siliconized borosilicate test tubes. Cells were then incubatedat 4° C. for 1 h with 40 pmol/L ¹²⁵I-VIP (2000 Ci/mmol) in the absenceor presence of competitor TP3654 with a known quantity, ranging from10⁻¹⁰ mol/L to 10⁻⁵ mol/L. Cells were then centrifuged and washed twicewith 1 mL 50 mmol/L Tris×HCl buffer (pH 7.5) at 4° C.; radioactivitybound to the cells was measured using a Packard 5000 series autogammacounter (Packard Instrument Corp., Meriden, Conn.). Similar assays wereperformed in triplicate in which VIP₂₈, instead of TP3654, was used as acompetitor. Using the Munsen National Institutes of Health (NIH)ligand-binding program (Bethesda, Md.), binding curves were plotted andhalf-maximal inhibitory concentrations (IC₅₀) were determined.

Tissue Distribution Studies With ^(99m)Tc-TP3654.

The ability of ^(99M)Tc-TP3654 to detect human colorectal carcinoma wasexamined in nude mice.

Human colorectal cancer cells LS 174T (ATCC) were grown in culture and5×10⁶ viable cells were implanted intramuscularly in the right thighs ofathymic NCr nude mice that weighed 20-25 g. Tumors were grown to no morethan 1 cm in diameter. Each animal received approximately 700 •Cl of^(99m)Tc-TP3654 (1450 Ci/mmol) in 200 •I, of saline through a lateraltail vein. Exact activity injected was determined by measuring thesyringe, before and after injection, in a calibrated ionization chamber(Capintech, Ramsey, N.J.). A suitable standard of ^(99m)Tc was preparedat the time of injection.

At 4 or 24 h later, animals were killed and imaged using a GE STARCAMgamma camera (Milwaukee, Wis.) equipped with a low-energy parallel-holecollimator and a dedicated computer. Tissues were dissected and weighed,and associated radioactivity was counted (Packard 5000 series gammacounter), along with a standard, in duplicate. Results were calculatedas percentage injected dose per gram of tissue (% ID/g) and wereanalyzed by using Student t test.

With ¹²⁵I-VIP

Approximately 1 •Ci ¹²⁵I-VIP (2000 Ci/mmol) was diluted to 200 •L in0.05 mol/L phosphate buffer at pH 6.5; 2 •g VIP₂₈ was added as a carrierso that the quantity of VIP injected per animal would be the same as for^(99m)Tc-TP3654. A tissue distribution study was performed in a mannersimilar to that for ^(99m)Tc-TP3654. These data served as positivecontrols against which ^(99m)Tc-TP3654 data were compared.

With 99-Tc-G(D)AGG Aba.

G(D)AGG-Aba, the chelating moiety and the spacer, was labeled with^(99m)Tc using the same procedure as for ^(99m)Tc-TP3654. The HPLCretention time for a single-peak ^(99m)Tc-G(D)AGG-Aba was 6.9 min,distinctly different from the 11.8 min required for ^(99m)Tc-TP3654. Foranimal tissue distribution studies, a protocol similar to that for^(99m)Tc-TP3654 was followed. Although G(D)AGG-Aba has considerablysmaller amino acid residues than TP3654, these data were importantbecause they helped determine whether any cleaving of^(99m)Tc-G(D)AGG-Aba had occurred by metabolic interaction with^(99m)Tc-TP3654.

Receptor Blocking

Unlabeled VIP-28 or TP3654 (50 •g intravenously) was injected into eachof the five LS 174T tumor-bearing nude mice; ^(99m)Tc-TP3654 (1450Ci/mmol; 700 •Ci intravenously) was administered 30 min later. Theamount of VIP₂₈ for 25-g mice (50 •g) was chosen randomly but with theknowledge that a larger amount would be toxic and the assumption that asmaller amount would block only a few receptors that would not besignificantly effective in decreasing ^(99m)Tc-7P3654 uptake. Mice werekilled 24 h later, and percentage injected dose per gram of tissues wasdetermined. Data were compared to 24 h distribution in mice (n=5) given700 •Ci ^(99m)Tc-TP3654 but not pretreated with unlabeled VIP or TP3654.

Blood Clearance in Rats

Three Sprague-Dawley rats, each weighing approximately 250 g, wereinjected through a lateral tail vein with 1 mCi ^(99m)TcTP3654 andserial blood samples were drawn, in triplicate, through the otherlateral tail vein at 1, 5, 10, 15 and 30 min and then at 1, 2, 4, 6, 18and 24 h. Blood samples were weighed, radioactivity was counted with astandard ^(99m)Tc solution prepared at the time of injection and thepercentage injected dose per gram of blood was plotted as a function oftime.

Results

The theoretical molecular weight as computed forVIP-Aba-Gly-Gly-(D)-Ala-Gly was 3654.32, whereas the observed molecularweight was 3654.48. In this analog, now called TP3654, no impuritieswere detected by either HPLC or Sciex APZ ion-spray mass spectrometry.The preparation of ^(99m)Tc-TP3654 and its proposed structure arepresented schematically in FIG. 1. The yield of ^(99m)Tc-TP3654 wasquantitative. The HPLC elution profile (FIG. 2) indicated that, unlikeMAG₃-VIP or CPTA-VIP, the ^(99m)Tc-TP3654 preparations were eluted in asingle peak (Rt=11.8 min) and that the radiochemical yield was >99%. TheHPLC analysis (free ^(99m)Tc Rt=3.8 min), also indicated that theproduct was stable for up to 24 h when it was stored at 22° C. and whenchallenged with 100 molar excess of DTPA, HSA or cysteine for 5 h at 37°C. However, at 24 h of incubation, approximately 11% of theradioactivity was displaced. Colloid content at all times was <5%.

The yields for ¹²⁵I-VIP and ¹²⁵I-TP3654 were approximately 90% and theretention times for mono-iodo and di-iodo products were 19.3 min and20.1 min, respectively. The specific activity of the mono-iodo product,free of unlabeled peptide, was approximately 1000 Ci/mmol.

The results of IAS smooth muscle tissue assays, which indicated thatTP3654 was able to exert 95% relaxivity at 10⁻⁶ mol/L, are shown in FIG.3. At this concentration, relaxivity by VIP₂₈ was 80% and relaxivity byCPTA-VIP was 35%. These data confirm previous findings that thebiological activity of CPTA-VIP was impaired and that the VEP analogTP3654 was as biologically active as VIP₂₈. These data corroborated withthe results of experiments in which TP3654 and VIP28 displaced the¹²⁵I-VIP binding of receptors expressed on human colorectal carcinomacells (HT29). These data are given in FIG. 4. The IC₅₀ values, asdetermined by the NIH ligand-binding program, for both TP3654 and VIP₂₈were approximately 15 nmol/L. These values were consistent with thosepreviously reported for VIP₂₈.

In Vivo Evaluation

In Table 1, the tissue distribution data for 4 and 24 h of^(99m)Tc-TP3654 and ¹²⁵I-VIP are given together with the 24 h tissuedistribution of ^(99m)Tc-G(D)AGG Aba. The distribution of^(99m)Tc-G(D)AGG Aba served as a reference and helped to ascertain thatthe distribution of ^(99m)Tc-TP3654 was distinctly different from thatof the ^(99m)Tc-chelating moiety. Data indicate that ^(99m)Tc-TP3654cleared by renal excretion with 18.99±3.75% ID/g at 4 h and 3.54±0.4%ID/g at 24 h. The liver uptake at these time points was 1. 12±0.08% ID/gand 0.33±0.04% ID/g, respectively, followed by the tumor uptake of0.24±0.08% ID/g and 0.23±0.13% ID/g, respectively. At 24 h afterinjection, ^(99m)Tc-TP3654 activity declined significantly in all organs(P<0.01) except the VIP receptor-rich tumor and the lungs (P=0.84 andP=0.78, respectively).

TABLE 1 Biodistribution (% ID/g + SD) of ^(99m)Tc-TP3654, ¹²⁵I-VIP and^(99m)Tc-G(D)AGG-Aba at 4 and 24 h Postinjection in Nude Mice BearingLS174T Colorectal Tumors (n = 5) ^(99m)Tc-TP3654 ¹²⁵I-VIP^(99m)Tc-G(D)AGG-Aba Tissue 4 h*^(†) 24 h*^(‡§) 4 h^(†‡) 24 h^(‡) 24h^(§) Muscle 0.09 ± 0.01 0.04 ± 0.01 3.32 ± 0.54 0.16 ± 0.03  0.01 ±<0+01 Small Intestine 0.18 ± 0.05 0.05 ± 0.01 2.12 ± 0.16 0.09 ± 0.010.03 ± 0.01 Heart 0.10 ± 0.00 0.06 ± 0.01 1.65 ± 0.07 0.09 ± 0.03   0.01± <0.01 Lung 0.17 ± 0.01 0.16 ± 0.09 3.98 ± 2.17 0.19 ± 0.10   0.03 ±<0.01 Blood 0.21 ± 0.02 0.12 ± 0.02 3.40 ± 0.55 0.07 ± 0.01   0.03 ±<0.01 Spleen 0.19 ± 0.05 0.11 ± 0.02 2.13 ± 0.36 0.12 ± 0.05 0.06 ± 0.03Kidney 18.99 ± 3.75  3.52 ± .040 2.46 ± 0.42 0.26 ± 0.03 0.20 ± 0.02Liver 1.12 ± 0.08 0.33 ± 0.04 1.98 ± 0.29 0.16 ± 0.03 0.16 ± 0.02 Tumor0.24 ± 0.08 0.23 ± 0.13 2.15 ± 0.36 0.06 ± 0.08   0.03 ± <0.01 TIM ratio2.73 ± 1.09 6.28 ± 3.09 0.54 ± 0.39 0.38 ± 0.56 1.88 ± 0.36 T/B ratio1.16 ± 0.29 1.98 ± 1.44 0.65 ± 0.16 0.88 ± 0.16 0.77 ± 0.03 *Pvalues for4 h and 24 h ^(99m)Tc-TP3654 are ≦ 0.01 for all tissues except for lung(P = 0.78), tumor (P = 0.84) and T/B ratio (P = 0.25) ^(†)Pvalues for 4h ^(99m)Tc-TP3654 and 4 h ¹²⁵I-VIP are ≦0.01 for all tissues ^(‡)Pvaluesfor 24 h ^(99m)Tc-TP3654 and 24 h ¹²⁵I-VIP are ≦0.01 for all tissuesexcept the heart (P = 0.11) lung (P = 0.55), spleen (P = 0.02) and T/Bratio (P = 0.02). ^(§)Pvalues for 24 h ^(99m)Tc-TP3654 and^(99m)Tc-G(D)AGG-Aba are ≦0.01 for all tissues except for the T/B ratio(P = 0.02). % ID/g = percentage injected dose per gram; T/M =tumor-to-muscle; T/B tumor-to-blood.

Although the tumor uptake was quantitatively low and remained unchanged,the tumor-to-blood and tumor-to-muscle ratios improved significantly(P<0.01) at 24 h after injection. The tumor uptake was nearly four timeshigher than that for ¹²⁵I-VIP at 24 h after injection (P<0.01). As thetime after injection elapsed, ¹²⁵I-VIP radioactivity in all tissues,including the tumor and lungs, declined, indicating deiodination fromVIP. At 24 h after injection, the ^(99m)TcG(D)AGG Aba was significantlylower in all tissues than that of ^(99m)Tc-TP3654 or ¹²⁵I-VIP.

A composite of posterior gamma-camera images of nude mice bearing humancolorectal tumor (LS174T), obtained with ^(99m)Tc-TP3654 and^(99m)TC-G(D)AGG-Aba, are given in FIG. 5. The image with^(99m)Tc-TP3654 (left) is distinctly different from the one with^(99m)Tc-G(D)AGG-Aba (right) and delineates the tumor and both kidneys.

Results given in Table 2 indicate that in mice treated with unlabeledTP3654 or VIP, the uptake of ^(99m)Tc-TP3654 decreased in all VIPreceptor-rich tissues except the kidneys. The uptake of ^(99m)Tc-TP3654in the spleen, liver, muscle and intestine in untreated mice was alreadylow, and even though it was decreased, it was not statisticallysignificantly different after VIP or TP3654 treatment (P=0.07, P=0.07,P=0.42 and P=0.48, respectively). Statistically significant decreaseswere observed in the heart, lungs, blood and tumor (P<0.01, P<0.02,P<0.01 and P<0.01, respectively). A similar trend was also apparent whenmice were treated with unlabeled TP3654. Particularly encouraging wasthe decreased uptake in the tumor and lungs, which suggested areceptor-blocking phenomenon and indicated that the uptake of^(99m)Tc-TP3654 was receptor specific. The increased kidney uptake mayhave been due to increased metabolites or to any pharmacological effectsthat may have been exerted by 50 •g VIP or TP3654. The blood clearancecurve given in FIG. 6 was biphasic; the •−t 1_(/2) was approximately 5min and the •−t_(1/2) was approximately 120 min. The •−t_(1/2) of 5 minfor ^(99m)Tc-TP3654 obtained in this study was similar to that reportedby Bolin et al. The results suggest that the TP3654, the VIP₂₈ analog,has retained the receptor specificity of VlP₂₈, which does not changewhen TP3654 is labeled with ^(99m)Tc.

TABLE 2 Tissue Distribution % ID/g ± SD) of ^(99m)Tc-TP3654 at 24 h inNude Mice Bearing Human Colorectal Tumors LS174T and Given Intravenously50 μg TP3654 or 50 μg VIP, 30 min Before the Administration of^(99m)Tc-TP3654 (n = 5) Tissue ^(99m)Tc-TP3654 TP3654 Pvalues* VIPPvalues^(†) Muscle 0.04 ± 0.01 0.04 ± 0.02 0.28   0.03 + <0.01 0.42Small intestine 0.05 ± 0.01 0.07 ± 0.02 0.13 0.04 ± 0.01 0.48 Heart 0.06± 0.01 0.04 ± 0.00 <0.01   0.02 ± <0.01 <0.01 Lung 0.16 ± 0.09 0.09 ±0.03 0.08 0.05 ± 0.01 0.02 Blood 0.12 ± 0.02 0.04 ± 0.01 <0.01   0.04 ±<0.00 <0.01 Spleen 0.11 ± 0.02 0.16 ± 0.01 <0.01 0.07 ± 0.01 0.07 Kidney3.52 ± 0.40 5.45 ± 1.48 0.04 12.98 ± 2.11  <0.01 Liver 0.33 ± 0.04 0.52± 0.03 <0.01 0.25 ± 0.06 0.07 Tumor 0.23 ± 0.13 0.09 ± 0.02 <0.01 0.07 ±0.03 <0.01 T/M ratio 6.28 ± 3.09 2.35 ± 1.01 0.02 2.45 ± 2.52 0.01 T/Bratio 1.98 ± 1.44 2.02 ± 0.52 0.05 1.54 ± 0.22 0.21 *Pvalues for 24 h^(99m)Tc-TP3654 in mice without and with treatment of 50 μg TP3654.^(†)Pvalues for 24 h ^(99m)Tc-TP3654 in mice without and with treatmentof 50 μg VIP. % ID/g = percentage injected dose per gram; VIP =vasoactive intestinal peptide; T/M = tumor-to-muscle; T/B = tumor toblood

Discussion

¹¹¹In-(DTPA-D-Phe¹)octreotide has been identified as a useful agent indiagnosis, prognosis and treatment of cancers and has stimulatedincreasing interest in inherent capabilities of radiolabeledreceptor-specific peptides for use in nuclear medicine. Through theoriginal work of Virgolini et al. and Reubi et al., another neuropeptide(VIP₂₈) has emerged as a highly potent compound for use in diagnosticimaging. VIP1 and VIP2 receptor subtypes are expressed in higherdensities and on more kinds of tumors than somatostatin. These data,together with the excellent physical characteristics of ^(99m)Tc,prompted the undertaking of the task of preparing ^(99m)Tc-VIP andevaluating it for functional integrity.

Earlier work, in which VIP was labeled with ^(99m)Tc by conjugating withBFCAs to His¹ at the amino terminus of VIP, met with difficulty:multiple radioactive species were formed and a significant amount ofbiological activity was lost. The current approach has eliminated thesedrawbacks and offers several advantages.

VIP₂₈, modified at Asn²⁸ by providing a tetrapeptide for strongchelation of ^(9m-)Tc, results in a single radioactive compound withquantitative yield and without compromising on the biologicalcharacteristics of VIP₂₈. Using this configuration, TP3654 has beenlabeled with stable rhenium for nuclear magnetic resonance (NMR) andcomputer modeling studies. This hybrid peptide approach eliminates thelengthy and frequently inefficacious procedures for synthesizing andconjugating BFCAs, blocking and deblocking certain functional groups andthe laborious purifying and characterizing of conjugated peptide. Usingthe same approach, a straight-chain hexapeptide, a straight-chainheptapeptide and a cyclized octapeptide have been labeled with ^(99m)Tc.The Aba spacer eliminates any steric hindrance from the chelating moietyand helps preserve the biological activity of the peptide. The chelatingmoiety can be added to either the carboxy or the amino terminus asneeded. Other amino acid combinations to provide the N₄ configurationalso can be chosen. The IC₅₀ values for VIP₂₈ and TP3654 as determinedby data shown in FIG. 4 are 15 nmol/L and are consistent with thosepreviously reported for VIP₂₈. Even more encouraging are the results ofthe functional IAS assay, shown in FIG. 3. This assay was based ontissue relaxivity, a true functional characteristic of VIP₂₈, the andprovides confidence in use of TP3654. The VIP receptor density for humancolorectal cell line LS 174T is not precisely known and may vary fromtumor to tumor. Nonetheless, tumors were clearly delineated with^(99m)Tc-TP3654 but not with ^(99m)Tc-G(D)AGG, the chelate. VIPreceptors are ubiquitous on normal tissues, although their density maybe lower than on the malignant cells. Consistently, a large proportionof ^(99m)Tc-TP3654 was observed in all tissues examined at 4 h afterinjection (Table 1). At 24 h after injection, this radioactivitydecreased in all tissues except in the lungs (P=0.78) and in the tumor(P=0.84). As a result, tumor-to-muscle ratios increased significantly(P=0.04) as the time after injection was prolonged, whereastumor-to-blood ratios improved only marginally.

The absolute tumor uptake was 0.23±0.13% ID/g, which is considerablyless than that with radiolabeled monoclonal antibodies. However, it wassignificantly greater than ¹²⁵I-VIP (0.06±0.08% ID/g; P=0.01). Such lowtumor uptakes of other radiolabeled receptor specific peptides have alsobeen observed in mice (0.08% ID/g), in rats (0.04% ID/g) and in humans(0.05% ID/g). In the clinical images, the lung uptake, although notquantified, was very high. In 25-g mice (Table 1), the lung uptake of^(99m)Tc-TP3654 at 4 h after injection was 0.17±0.01% ID/g, whereas in250-g rats, the lung uptake at the same time averaged 0.73±0.12% ID/g.

Although the lung uptake was the same as the tumor in mice on a per unitweight basis (Table 1), lungs were not delineated by scintigraphicimaging (FIG. 5), probably because of their low weight and wide areaspread. The maximum uptake of ^(99m)Tc-TP3654 was in the kidneys.However, this was not contributed by ^(99m)Tc-G(D)AGG-Aba (0.2±0.02%ID/g; P=0.01) (Table 1), indicating that the chelating moiety was intactand that the renal uptake is caused by the uptake of ^(99m)Tc-TP3654 orits metabolic products.

Conclusion

A new hybrid peptide technique has been developed for labeling peptideswith ^(99m)Tc. It is applicable to labeling any peptide with^(99m)Tc-short or long, cyclized or not—and also is applicable topeptide labeling with radionuclides of rhenium. This method is simpleand efficient and has several advantages over conventional bifunctionalchelating methods.

Preparation of a Kit for Preparing a Tumor Imaging Agent

A preferred kit formulation is one which will label the composition offormula (I) or (II) instantaneously at room temperature, permits greaterthan 95% of the added radiolabel to bind to the composition, and has ashelf life of six months or longer.

The kit will be comprised of a carrier and a vial of a composition offormula (I) or (II), stannous chloride, and buffers. The vial will becapable of holding lyophilized reagents as appropriate and will besealed under nitrogen. Stabilizing agents or anti-oxidants such as EDTAor ascorbic acid may be added to the reagents to increase the shelf lifeof a kit. Additional vials may contain appropriate reagents including,but not limited to, a buffer, such as 0.05 M phosphate buffer.Antioxidant agents may also be included to prevent oxidation of Sn²⁺ toSn⁴⁺. Sn²⁺ is necessary for reduction of Tc⁷⁺ to lower oxidation statesrequired for chelation.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

3 1 28 PRT Artificial Sequence Synthetic Peptide 1 His Ser Asp Ala ValPhe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val LysLys Tyr Leu Asn Ser Ile Leu Asn 20 25 2 33 PRT Artificial SequenceSynthetic Peptide 2 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg LeuArg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu AsnXaa Gly Gly Ala 20 25 30 Gly 3 4 PRT Artificial Sequence SyntheticPeptide 3 Gly Ala Gly Gly 1

What is claimed:
 1. A method for imaging tumors in a mammal, said methodcomprising: (a) injecting into said mammal in need of such imaging aneffective diagnostic amount of a composition comprising a pharmaceuticalcarrier and a compound having formula I or II: M-Z-X₁—P—X₂  (I)X₁—P—X₂-Z-M  (II) wherein: M is a four-amino acid radiolabeling moietycomplexed to a radionuclide in an N₄ configuration; Z is 4-aminobutyricacid; X₁ is from zero to twenty natural or synthetic amino acids; P is apeptide having the amino acid sequenceHis-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn(SEQ ID NO: 1), or an analog or fragment thereof, wherein said analog orfragment retains the biological activity of SEQ.ID.NO: 1; and X₂ is fromzero to twenty natural or synthetic amino acids; and (b) scanning saidmammal using radioscintigraphic imaging apparatus to image tumors insaid mammal.
 2. The method according to claim 1 wherein the imagedtumors comprise colorectal carcinoma.
 3. The method according to claim 1wherein the imaged tumors comprise liver metastases.
 4. The methodaccording to claim 1 wherein the imaged tumors comprise pancreaticcarcinoma.
 5. A method for detecting tumors in a mammal comprising (a)administering to the mammal an effective diagnostic amount of a compoundhaving formula I or II: M-Z-X₁—P—X₂  (I) X₁—P—X₂-Z-M  (II) wherein: M isa four-amino acid radiolabeling moiety complexed to a radionuclide in anN₄ configuration; Z is 4-aminobutyric acid; X₁ is from zero to twentynatural or synthetic amino acids; P is a peptide having the amino acidsequenceHis-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn(SEQ ID NO: 1), or an analog or fragment thereof, wherein said analog orfragment retains the biological activity of SEQ.ID.NO: 1; and X₂ is fromzero to twenty natural or synthetic amino acids; and (b) detectingradiolabel localized at the tumor in the mammal.
 6. The method accordingto claim 5 wherein the detected tumors comprise colorectal carcinoma. 7.The method according to claim 5 wherein the detected tumors compriseliver metastases.
 8. The method according to claim 5 wherein thedetected tumors comprise pancreatic carcinoma.